Vibrio cholerae modulates the
immune defense of human gut
mucosa
Aziz Bitar
Department of Clinical Microbiology, Section of Infection and Immunology
This work is protected by the Swedish Copyright Legislation (Act 1960:729) Dissertation for PhD
ISBN: 978-91-7601-881-1 ISSN: 0346-6612
New Series Number 1962
Front cover: Electron micrographs of small intestinal epithelium (upper left), V. cholerae bacteria (upper right and middle left), T84 tight monolayer (middle right and lower right) and outer membrane vesicles of V. cholerae (lower left). (With courtesy of Professors Marie-Louise Hammarström and Sun Nyunt Wai). Electronic version available at: http://umu.diva-portal.org/
Printed by: UmU-tryckservice, Umeå University Umeå, Sweden 2018
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Table of Contents
ABSTRACT ... iii
Populärvetenskaplig sammanfattning ... v
Abbreviations ... vii
PAPERS IN THE THESIS ... ix
INTRODUCTION ... 1
1. BACKGROUND ... 2
1.1 Overview of the immune system ... 2
1.2 Adaptive immunity ... 2
1.3 Innate Immunity ... 4
1.3.1 Cellular components of the innate immunity ... 4
1.4 Mucosal immunity and the intestine... 7
1.4.1 General structure and cellular composition of the intestine ... 7
1.4.2 Tissues of the mucosal immune system and intestinal immune cells ... 11
1.6 Pattern recognition receptors ... 13
1.6.1 Toll-like receptors ... 13
1.6.2 Other families of pattern recognition receptors ... 14
1.6.3 Pattern recognition receptors in human intestine... 15
1.7 Cytokines ... 17
1.7.1 Interleukin-1β and Interleukin-18 ... 18
1.7.2 Tumor Necrosis Factor−α ... 19
1.7.3 Chemokines ... 19
1.9 MicroRNA... 21
1.9.1 Biogenesis, maturation and function of microRNA ... 22
1.9.2 MicroRNA in innate responses to pathogens ... 23
1.10 Bacterial interactions with the gastrointestinal tract ... 25
1.11 Vibrio cholerae... 25
1.11.1 O1 and O139-associated virulence factors... 27
1.11.2 Non-O1 and non-O139 strains and associated virulence factors... 28
1.12 Bacterial outer membrane vesicles ... 30
2. AIMS ... 33
3. METHODOLOGICAL CONSIDERATIONS ... 34
3.1 Clinical material ... 34
3.2 Polarized tight monolayers of human intestinal epithelial cells ... 34
3.3 Bacterial work and effects on target cells ...35
3.4 TER measurement techniques ... 36
3.5 Gene expression analysis of total RNA using real-time qRT-PCR ... 37
3.5.1 Determination of expression levels of target-genes, cytokine and chemokine mRNAs by using real-time qRT-PCR ... 37
3.5.2 Determination of expression levels of microRNAs using real-time qRT-PCR ... 38
3.5.3 Housekeeping genes for stable normalization ... 38
3.5.3 Gene expression analysis using genome-wide hybridization bead array ... 39
3.6 In situ miRNA hybridization ... 40
3.7 Determination of amounts of cytokine protein ... 40
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3.8.1 Nanotracking analysis ... 41
4. RESULTS AND DISCUSSION ... 42
4.1 Rationale of project ... 42
4.2 Innate epithelial responses to secreted Vibrio cholerae cytolysin and PrtV of Vibrio cholerae ... 42
4.2.1 Effects of culture supernatant of V. cholerae on epithelial cells ... 42
4.2.2 Role of Vibrio cholerae cytolysin ... 44
4.2.3 Post-translational modulation of inflammation by PrtV ... 46
4.2.4 V. cholerae cytolysin induces interleukin-1 receptor activated kinase 2 ... 47
4.3 Cytokines and microRNA expression in small intestinal mucosa during acute Vibrio cholerae O1 infection ... 47
4.3.1 Analysis of cytokine mRNA expression ... 47
4.3.2 Induction of regulatory microRNA in the intestinal epithelium ... 48
4.3.3 Analysis of targets for miR-146a in the intestinal mucosa ... 50
4.3.4 In vitro analysis of V. cholerae and its secreted factors ... 50
4.4 Effects of released outer membrane vesicles ... 51
4.4.1 Tightening of epithelia ... 51
4.4.2 Modulation of cytokine responses ... 52
4.4.2 Induction of regulatory microRNA-146a ... 53
4.5 Vibrio cholerae infection and the role of microRNA and released components – summary of results and hypothesis ... 54
5. CONCLUSIONS ... 56
6. ACKNOWLEDGEMENTS ... 58
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ABSTRACT
The key function of innate immunity is to sense danger signals and initiate effective responses as a defense mechanism against pathogens. Simultaneously, effector responses must be regulated to avoid excessive inflammation with resulting tissue damage. microRNAs (miRNAs), are small endogenous molecules, that has recently gained attention as important regulatory elements in the human inflammation cascade. The control over host miRNA expression may represent a previously uncharacterized molecular strategy exploited by pathogens to mitigate innate host cell responses.
Vibrio cholerae is a Gram-negative bacterium that colonizes the human small intestine and causes life-threatening secretory diarrhea, essentially mediated by cholera toxin (CT).It is considered a non-invasive pathogen and does not cause clinical inflammation. Still, cholera is associated with inflammatory changes of the small intestine. Furthermore, CT-negative strains of V. cholerae cause gastroenteritis and are associated with extra-intestinal manifestations, suggesting that other virulence factors than CT are also involved in the pathogenesis.
The innate immune response to V. cholerae is poorly investigated and the potential role of miRNA in cholera had not been studied before. Therefore, this thesis explores the role of intestinal epithelial cells in response to V. cholerae infection with a focus on regulatory miRNA as a potential contributor to the pathogenesis. The in vivo material was small intestinal biopsies from patients suffering from V. cholerae infection. As an in vitro model for V. cholerae attack on intestinal epithelium, we used tight monolayers of T84 cells infected with V. cholerae and their released factors. We analyzed changes in levels of cytokines, immunomodulatory microRNA and their target genes.
We showed that miRNA-146a and miRNA-155 reached significantly elevated levels in the intestinal mucosa at acute stages of disease in V. cholerae infected patients and declined to normal levels at the convalescent stage. Low-grade inflammation was identified at the acute stage of V. cholerae infection, which correlated with elevated levels of regulatory miRNA. Furthermore, outer membrane vesicles (OMVs) released by the bacteria were shown to induce miR-146a and live bacteria induced miR-155 in intestinal epithelial cells. In addition, OMVs decreased epithelial permeability and caused mRNA suppression of pro-inflammatory cytokines, including immune cell attractant IL-8 and CLL20, and the inflammasome markers IL-1β and IL-18. These results propose that V. cholerae regulates the host expression of miRNA during infection and may set the threshold for activation of the intestinal epithelium.
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Moreover, we showed that V. cholerae also harbors inflammatory-inducing capabilities, by secreting a pore-forming toxin, Vibrio cholerae cytolysin (VCC). By using genetically modified strains as well as soluble protein challenge experiments, VCC was found solely responsible for the increased epithelial permeability and induction of several pro-inflammatory cytokines in intestinal epithelial cells. In contrast to OMVs, VCC displayed strong upregulation of the pro-inflammatory cytokines IL-8, TNF-α, CCL20 and IL-1β and IRAK2, a key signaling molecule in the IL-1 inflammasome pathway. This suggest that VCC is an important virulence factor in the V. cholerae pathogenesis, particularly in CT-negative strains. Furthermore, we showed that the bacterium could control the inflammatory actions of VCC by secreting the PrtV protease, which degraded VCC and consequently abolished inflammation.
In summary, we showed that V. cholerae harbors immunomodulating capabilities, both at the gene level, through induction of host regulatory miRNA, and at the protein level, through secretion of VCC and PrtV. These strategies may be relevant for V. cholerae to promote survival in the gut and cause successful infections in the human host.
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Populärvetenskaplig sammanfattning
Många sjukdomsframkallande tarmbakterier använder sig av olika strategier för att reglera och undvika immunförsvaret i tarmen. För bakterien Vibrio cholerae, även kallad kolerabakterien, vilken utgör ett stort hot mot människors liv och hälsa världen över, är dock dessa immunreglerande mekanismer inte särskilt välstuderade. För att tillhandahålla bättre förebyggande och terapeutiska åtgärder är det viktigt att fördjupa förståelsen kring dessa mekanismer.
Tarmslemhinnan, eller tarmepitelet, som täcker tarmen insida, är en viktig del av kroppens immunförsvar och har en svår uppgift. Den måste tolerera den godartade tarmfloran och samtidigt försvara sig mot sjukdomsframkallande bakterier. Att reglera graden av inflammation, kroppens svar på infektion, är viktigt för att inte orsaka vävnadsskador i tarmen. På senare tid har mikroRNA (miRNA), små bitar av RNA som styr genuttryck, uppmärksammats som viktiga faktorer i regleringen av den inflammatoriska reaktionen. Det har också visats att vissa sjukdomsframkallande bakterier kan utnyttja miRNAs inflammations-hämmande egenskaper för att undkomma kroppens immunförsvar.
Bakterien V. cholerae infekterar tarmen och orsakar stora mängder vattnigt diarré som snabbt kan leda till livsfarlig uttorkning om inte behandling sätts in tidigt. Bakterien utsöndrar olika komponenter, till exempel toxiner och vesiklar, som deltar i sjukdomsförloppen. Toxiner är ett slags gift, och vesiklar är små membranomgivna bubblor som skickas iväg av bakterien och innehåller faktorer som kan påverka mottagarcellerna i tarmepitelet.
Den kolerastam som orsakar alla större epidemier och pandemier verkar främst genom det välstuderade så kallade koleratoxinet. Idag förekommer dock även andra kolerastammar som trots avsaknad av toxinet, ändå orsakar lokala utbrott av diarrésjukdom med inflammatoriska inslag. De sjukdomsframkallande mekanismerna hos dessa stammar är fortfarande inte kända. Det är heller inte kartlagt huruvida V. cholerae kan uttrycka miRNA i tunntarmsslemhinnan under infektionsförloppet. Det är heller inte känt hur vesiklar utsöndrade av V. cholerae påverkar immunförsvaret i tarmen.
Syftet med denna avhandling är att förstå reaktionerna i tarmsepitelet under V. cholerae-infektion, inklusive de utsöndrade bakteriella komponenternas påverkan på tarmepitelet.
I studie I har vi använt av oss av ett framodlat tunntarmsepitel, som har liknande egenskaper som det normala tarmepitelet. Genom denna modell har vi kunnat studera enskilda bakteriella komponenters påverkan på tarmepitelet. Vi
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visar att V. cholerae kan utsöndra en känd faktor kallad V. cholerae cytolysin (VCC), som i tunntarmen orsakar inflammation och ökad genomsläpplighet i epitelet. Denna reaktion innefattar en uppreglering av ett flertal inflammatoriska markörer, som kan signalera för immunceller att komma till infektionsområdet i tarmen. Vidare visar vi att bakterien kan utsöndra en annan faktor PrtV som bryter ner VCC och på så vis kan påverka mängden inflammation till sin fördel.
I studie II och III har vi, förutom det framodlade tunntarmsepitelet, också använt oss av tunntarmsbiopsier från patienter i olika stadier av kolerasjukdom. Genom dessa två metoder har vi studerat uttrycket av miRNA och hur immunförsvaret i tarmen påverkas under sjukdomsförloppet. I studie II visar vi att förhöjda nivåer av miRNA-146a och miRNA-155 återfinns i tarmepitelet hos patienter vid akut kolerainfektion, men sjunker sedan till normala nivåer vid tillfrisknandet. Fyndet av miRNA korrelerar väl med ett minskat inflammatoriskt påslag i tunntarmsslemhinnan. Vi fann vidare i studie III att inte bara bakterien, utan även utsöndrade vesiklar kan få värdcellerna att uttrycka miR-146a som därmed verkar hämmande för det inflammatoriska påslaget. Via vesiklar kan bakterien således dämpa det inflammatoriska svaret utan att själv behöva vara i direktkontakt med värdcellerna i tarmen.
Sammanfattningsvis visar våra resultat hur V. cholerae både på gennivå, via att uttrycka miRNA hos värdcellen, och på proteinnivå genom att utsöndra VCC och PrtV kan orsaka och modulera graden av inflammation i tarmslemhinnan och därmed skapa optimala förhållanden för sin överlevnad.
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Abbreviations
ADCC Antibody-dependent-cell mediated cytotoxicity
AMP Antimicrobial peptide
APC Antigen-presenting cell
BCR B cell receptor
CD Cluster of differentiation
CTL Cytotoxic T-cell
CTX Cholera toxin
DAMP Danger-associated molecular pattern
DC Dendritic cell
FAE Follicle-associated epithelium
GALT Gut-associated lymphatic tissue
HBD Human β defensin
HLA Human leukocyte antigen
IBD Inflammatory bowel disease
IEC Intestinal epithelial cell
IFN Interferon
Ig Immunoglobulin
IL Interleukin
ILC Innate lymphoid cell
LI Large intestine
LP Lamina propria
LPL Lamina propria lymphocytes
LPS Lipopolysaccharide
LRR Leucin-rich repeat
MHC Major histocompatibility complex
MIC MHC class I chain related antigen
mRNA Messenger RNA
miRNA microRNA
MUC2 Mucin 2
NF-κB Nuclear factor kappa B
NLR NOD-like receptor
NK Natural killer cell
NKT Natural killer T cell
NOD Nucleotide oligomerization domain
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PCR Polymerase chain reaction
PP Peyer’s patch
PRR Pattern recognition receptor
PrtV Protease of Vibrio cholerae
qRT-PCR Real-time quantitative reverse transcriptase-PCR
RLR Retinoic acid-inducible gene (RIG)-I-like receptor
SI Small intestine
sIgA Secretory IgA
TCR T cell receptor
TFF Trefoil factor
TGF Transforming growth factor
Th T helper cells
TJ Tight junction
TLR Toll-like receptor
TNF Tumor necrosis factor
Treg Regulatory T cell
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PAPERS IN THE THESIS
This thesis is based on the following articles and manuscripts, which will be referred to in the text by Roman numerals (I-III).
I. Ou, G., Rompikuntal, P.K., Bitar, A., Lindmark, B., Vaitkevicius, K., Bhakdi, S., Wai, S. N., Hammarström, M-L. Vibrio cholerae cytolysin causes an inflammatory response in human intestinal epithelial cells that is modulated by the PrtV protease. PLoS One. 2009 Nov
12;4(11):e7806.
II. Bitar, A*, De, R*, Melgar, S., Aung, K.M., Rahman, A., Qadri, F., Wai,
S.N., Shirin, T¤, Hammarström¤, M-L.Induction of Immunomodulatory
miR-146a and miR-155 in Small Intestinal Epithelium of Vibrio cholerae Infected Patients at Acute Stage of Cholera. PLoS One. 2017 Mar 20;12(3): e0173817.
III. Bitar, A., Aung, K.M., Wai, S.N., Hammarström, M-L. Vibrio cholerae
derived outer membrane vesicles modulate the inflammatory response of human intestinal epithelial cells by inducing microRNA-146a
(Submitted)
*These authors contributed equally to this work ¤These authors shared senior authorship
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INTRODUCTION
The intestinal epithelium consists of a single layer of epithelial cells. It is constantly exposed to luminal antigens from food and the normal microflora and episodic infections by pathogens. The epithelium itself is an important and integrated compartment of the gut immune system. The powerful effector responses by the gut immune system must tolerate the commensal microflora and simultaneously avoid detrimental effects for the host.
Vibrio cholerae, a Gram-negative, small intestinal pathogen, infects the gut and causes severe life-threatening diarrhea. It was previously thought that cholera was solely a non-inflammatory and non-invasive disease. However, recent reports have provided evidence for inflammatory changes of the intestine during clinical infection. This was further illustrated by the fact that V. cholerae lacking the cholera toxin could still cause disease, such as gastroenteritis and extra-intestinal manifestations. These findings suggest that other virulence factors are involved in the pathogenesis. However, the nature of these inflammation-causing factor(s) in V. cholerae is largely unknown.
Micro(mi)RNAs are small endogenous RNAs that post-transcriptionally regulate eukaryotic gene expression. In addition to their involvement in a wide range of physiological conditions, miRNAs are increasingly implicated in the host cell response to bacterial pathogens. By gaining control over host miRNA, bacteria can modulate host responses and provide optimal conditions for successful infections.
In this thesis, in vitro and in vivo studies of V. cholerae infection are conducted and new insights are provided on how the bacterium can modulate host immune responses, at the post-transcriptional and post-translational level.
The following chapters give a brief background of the gut mucosal immune system, miRNA and V. cholerae before the discussion of the results obtained in this study.
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1. BACKGROUND
1.1 Overview of the immune system
The word immunity, from the Latin, immunis, meaning “exempt” (or free of burden), originates from the juridical concept ‘exception’. Although, it was not until the late nineteenth century in Europe, that the word became a medical term referring to the state of protection from infectious disease.
The human immune system provides a vital defense mechanism and is defined by three main functions: Firstly, the ability to sense and deploy effective defense reactions against invading microorganisms, such as bacteria, virus, fungi, protozoa and parasites. Secondly, the constant immune surveillance that recognizes and removes damaged or transformed cells and thus contributes to the prevention of tumor development. Thirdly, promoting homeostasis, an attribute that is mainly important at constantly exposed surface areas, such as mucosal sites, where the external and sometimes hostile environment encounters bodily tissues. In addition, the immune system must also regulate and tolerate the beneficial commensal microbes that are localized along mucosal tissues.
The immune system is traditionally divided into two branches, the innate and the adaptive, which are interconnected and collaborate to protect the host against pathogens.
1.2 Adaptive immunity
The adaptive immune system is traditionally defined by its three main characteristics: antigen specificity, immunological memory, and the ability to discriminate between self/non-self-antigens. In contrast to the rapid and unspecific innate immunity, adaptive responses take days to be activated following the first encounter with a specific pathogen. Adaptive immunity can be divided into a humoral and mediated immune responses. The cell-mediated responses are cell-mediated by specific T lymphocytes, T cells, and the humoral responses by antibodies derived from specific B lymphocytes, B cells. The specificity of the adaptive immune system is created by somatic rearrangements of the encoding genes that create enormous pools of T cells and B cells. Each have one specificity per cell that is mediated by T cell receptor (TCR) and B cell receptors (BCR), respectively. Antibodies are produced by plasma cells, which in turn are maturated from the specific B cells.
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The activation of adaptive immunity is based on recognition of exogenous and/or endogenous peptides presented by antigen-presenting cells (APCs), which harbor cell surface proteins of the major histocompatibility complex (MHC). MHC molecules can be divided into two classes: MHC class I and MHC class II. Most nucleated cells express MHC I class and therefore present peptides from endogenous antigens, such as viral and tumor antigens. MHC class II molecules present processed extracellular antigens to T cells and are normally expressed by professional APCs, like B cells, dendritic cells (DCs), and macrophages.
T cells originate from the bone marrow and mature in the thymes, where they learn to discriminate between self- and non-self-antigens through positive and negative selection processes. As the human thymus regress over time, other organs are also involved in T cells maturation, such as the intestinal mucosa1,2. T
cells recognize processed antigens by their TCRs. Based on the composition of TCR polypeptide chains, T cells can be divided into αβ T cells and γδ T cells that differ in their biology and functions. In association with TCR, T cells express surface marker, cluster of differentiation 3 (CD3), which is involved in the activation of intracellular signaling upon antigen recognition.
αβT cells are further classified based on expression of co-receptors CD4 or CD8. The CD8+ T cells recognize antigens in association with MHC class I molecules,
while CD4+ recognize antigens presented by MHC class II molecules. T cells
require two signals for activation: recognition of antigens presented on MHC I or II by the TCR/CD3 complex, and interaction with the co-stimulatory molecules presented on APCs. The most important co-stimulatory signals are mediated through CD28 and CD40L on T cells, which bind to CD80 and CD40 on APCs. Naïve T cells refer to T cells that have not yet encountered an antigen. T cells proliferate and differentiate into diverse types of effector T cells, depending on the stimuli and the cytokine milieu. CD8+ T cells can differentiate
into cytotoxic T (CTLs) cells that kill infected or transformed cells. Most CD4+ T
cells are T helper cells (Th) that assist other immune cells in immunologic processes and include different T cell subsets. For example, Th1, Th2 and Th17 are responsible for pathogen clearance and produce cytokines and other soluble mediators involved in efficient removal of their respective target pathogens. In addition, a regulatory T cell subset (Treg) is responsible for maintaining intestinal homeostasis by down-regulating the production of pro-inflammatory cytokines, and thus dampening inflammation responses.
The B cells differentiate in the bone marrow and they express the BCR, which is a membrane-bound immunoglobulin (Ig) with a unique antigen binding site.
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Each B cell has only one antigen-binding site i.e. specificity. The BCR is composed of two identical light and two identical heavy chains with variable V(D)J and constant (C) regions. The V(D)J region determines the antigen specificity and the C region determines the effector functions and antibody class. Naive B cells express BCR of IgM and IgD classes on their cell surfaces. Upon activation, which generally occur with the help of T cells, B cells differentiate into plasma cells, a differentiation process that induces changes in the constant region to IgG, IgA or IgE, a process called isotope switching. Antibodies have several important functions, such as toxin neutralization, prevention of adhesion of pathogens to mucosal surfaces, activation of the complement system, opsonization of microorganism for phagocytosis and involvement in antibody-dependent-cell-mediated-cytotoxicity (ADCC). Antibody producing plasma cells are usually short lived, but a fraction of these cells become memory cells3.
1.3 Innate Immunity
Innate, derived from the Latin, innātus, meaning inborn, refers to the immune response system that we are born with. It is an evolutionary ancient defense mechanism, found in plants, fungi, insects and other multicellular organism. Innate immunity constitutes the “first” line of host defense and controls the first step of immune response. Despite constant exposure to microorganisms, the innate immune systems keep the incidence of infectious diseases relatively low in the healthy human body. It senses and provides immediate protection against potential threats, without requiring previous exposure to pathogens.
Essential elements in innate immunity includes: provision of physical and biochemical barriers, sensing the presence of microbes and tissue damage, production of antimicrobial compounds, e.g. antimicrobial peptides (AMPs), lysosome, lactoferrin, reactive oxygen and nitrogen species, activation of the complement cascade, cytokines for activation and recruitment of immune cells to the site of an infection, and direct or indirect killing of microbes by immune cells that compromise the innate immunity.
The basis of recognition in innate immunity, resides in the ability to sense unique patterns that are common to most microbes, referred to as MAMP (microbe-associated molecular pattern), which are recognized by host sensors known as pattern recognition receptors (PRRs).
1.3.1 Cellular components of the innate immunity
The leucocytes involved in innate immunity include macrophages/monocytes, dendritic cells (DCs) granulocytes, unconventional T cells and innate lymphoid
cells (ILCs) and natural killer (NK) cells, which together are responsible for the rapid recognition and elimination of pathogens. In addition to pathogen
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removal, most cellular components of the innate immune system also serve as links to activate the adaptive immune system.
1.3.1.1 Monocytes are the largest cell of the leukocytes in term of size and originate from a precursor in the bone marrow. They enter the peripheral blood and migrate by the help of specific chemokine and adhesion receptors to sites of inflammation or infection and then mature to macrophages. Macrophages display important functions in both innate and adaptive immunity. They reside in almost all tissues and are believed to maintain tissue integrity. Further, they are usually the first immune cells to encounter foreign material, such as bacteria and soluble proteins. They can engulf and kill microbes and promote inflammation by producing cytokines and chemokines that activate and recruit other immune cells to the site of infections.
1.3.1.2 Dendritic cells (DCs) are potent APCs cells and serve as messengers between the innate and adaptive immune responses. Morphologically, long cytoplasmic processes that allow intimate contact with several immune cells, characterize the DCs. These cells can present antigens in the context of MHC class II molecules and express the surface molecules CD80 and CD40, which are accessory molecules required for T cell activation. DCs are found in all lymphoid organs and mucosal sites.
1.3.1.3 Granulocytes
Granulocytes are characterized by their cytoplasmic granules, which are secretory vesicles containing various antimicrobial factors. They are also called polymorphonuclear leukocytes (PMN) because of the varying shape of the nucleus, which is usually lobed into three segments. PMNs often refer to neutrophils, since this group is the most abundant of all granulocytes. Other groups of granulocytes are eosinophils, mast cells, and basophils.
Neutrophils comprise more than half of the circulating leukocytes and are usually the first immune cells to arrive at the site of infection or inflammation. They are recruited by a process called chemotaxis, meaning that they migrate towards a chemical gradient to substances such as interleukin-8 secreted by macrophages and epithelial cells at the site of infections. They eliminate pathogens by phagocytosis or by degranulation that releases soluble antimicrobial compounds such as defensins. Furthermore, they can form massive amounts of reactive oxygen and nitrogen species and other toxic molecules that destroy pathogens. They also generate neutrophil extracellular traps (NET), which are a network of composed neutrophil DNA that binds pathogens4.
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Eosinophils comprise a small part of circulating leucocytes. They are not phagocytic, but contain antimicrobial granules that are released upon activation and usually involved in defense against parasites5.
Mast cells are characterized by their content of numerous secretory granules, containing proteases, histamine, serotonin, heparin and chemokines. They express IgE receptors (FcεR), which bind antibodies of the IgE class. If appropriate antigen (often called allergen in this context) is bound to the receptor-bound IgE, cross-linking occurs which in turn leads to degranulation. Mast cells are associated with allergy and anaphylaxis, but are also involved in defense against pathogens, including parasitic infections.
Basophils are the smallest population of blood leukocytes and may functionally be considered as circulating form of mast cells. Basophils release effector molecules and vasodilating substances, (e.g. histamine), usually involved in allergen responses.
1.3.1.2 Unconventional T cells
Unconventional T cells share features of cells belonging to both innate and adaptive immunity. They primary recognize lipids and small-molecule metabolites. Further, they express a TCR, which generally has low diversity and respond rapidly upon activation. Among the most studied unconventional T cells are mucosal associated invariant T cells (MAIT), γδ T cells and NKT cells. MAIT cells are evolutionarily conserved T cells expressing CD8 and recognizing bacterial metabolites via the non-classical MHC class I- related molecule (MR1). They are primarily found in the circulation, but can also be found in the gut6.
γδ T cells constitute only a small proportion of the lymphocytes in human blood, although they are widespread within the intestinal epithelium7. They can
recognize a variety of antigens, such as small non-peptide ligands and larger protein antigens, such as MHC class I chain-related antigens A and B (MICA/MICB) expressed on stressed cells. γδ T cells attack target cells directly through degranulation of cytotoxic granules or indirectly through the activation of other immune cells.
Natural killer T cells (NKT cells) are a subset of lymphocytes that express TCR, but also other molecules that are characteristic of NK cells. They recognize glycolipid antigens presented by the non-classical MHC I molecule CD1d and are found with the highest frequency in the lamina propria (LP) of the small intestine8. Upon activation, they are involved in defense mechanisms against
tumours and infectious microbes, but can also suppress cell-mediated immune responses9.
7 1.3.1.4 Innate lymphocyte cells
Like unconventional T cells, the innate lymphocyte cells (ILCs) share features with cells of both the adaptive and innate immune system. One of their characteristics is the absence of TCR. ILCs are divided into three groups (ILC1, ILC2 and ILC3) based on cytokine-production profiles10. ILC1 consist of
IFN-γ-producing cells that play a major role in defense against viruses and intracellular bacteria. ILC2 produce cytokines associated with parasitic infections and allergy, while ILC3 are involved in lymphoid tissues development as well as maintaining the intestinal barrier integrity11.
1.3.1.5 NK cells are cytotoxic lymphocytes that are essential in innate immunity and kill target cells with low expression of MHC class I molecules (i.e. virus-infected cells or tumour cells). NK cells lack antigen-specific receptors and are not dependent on MHC for their activation. Instead, their cytotoxic activity is tightly regulated by stimulatory and inhibitory receptors on the cell surface12.
Antibodies attached to infected cells can be recognized by NK cells and trigger them to kill the infected cell through antibody-dependent cell-mediated cytotoxicity (ADCC).
1.4 Mucosal immunity and the intestine
The mucosal immune system consists of diffusely distributed immune cells and cells organized into lymphoid tissues as well as within epithelial cells. It is present in the gastrointestinal (GI), respiratory, and urogenital tracts. Together it constitutes the largest areas within the body that is in contact with the external environment. These surface areas separate the bodily tissues from pathogenic microbes and commensal flora, often by a single layer of epithelial cells. Functions associated with the mucosal immune system is mucus production, providing a physical barrier, ability to tolerate the normal microbiota and in the GI also food constituents. Constant peristalsis in the intestine may also be considered functions of this system.
1.4.1 General structure and cellular composition of the intestine
The intestine is the largest mucosal surface in the body, and therefore houses the largest reservoir of immune cells. It is divided into the small intestine (SI) and the large intestine (LI). The SI compromises about 80 % of the entire intestine and is about 8.5 m long in adult humans 13. The small intestine (SI) can
be further divided into three segments: the duodenum, which begins at the pylorus, and is followed by the jejunum, and the ileum. Ileum ends in the ileocecal valve, which is the entry point into the LI. The LI begins at the caecum, followed by the ascending colon, the transverse colon, the descending colon and the rectum.
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The SI plays a role in nutrient absorption and consists of villi, finger-like projections extending into the lumen, which increases the surface area for nutrition absorption. The base of each villus is surrounded by numerous epithelial invaginations, known as crypts of Lieberkühn. The LI, which is primarily involved in water absorption and elimination of undigested dietary components, lacks villous structures and has deeper crypts.
Histologically, four distinct layers can be distinguished along the whole intestine. These are: the mucosa, the submucosa, the muscularis externa and the serosa. The mucosa faces the gut lumen and can be further divided into the intestinal epithelium, the lamina propria (LP), and the muscular mucosa. Under the intestinal epithelium, is a layer of connective tissue, the LP. The LP supports the epithelium and is a compartment that houses different types of cell populations, most of them belonging to the immune system, such as T and B cells, plasma cells, macrophages, DCs and occasionally eosinophils. Additionally, connective tissue cells, such as fibroblast and smooth muscle cells are present in LP. A network of blood capillaries and lymphatic capillaries are also present in LP. The muscularis mucosae is a thin muscle layer separating the LP from the submucosa. The submucosa in turn is a layer of connective tissue harboring fibroblast, mast cells, blood- and lymphatic vessels and nerve fiber plexus. Under the submucosa reside layers of smooth muscle tissue, the muscularis externa, which play a role in the peristalsis. The fourth layer of the intestine wall is the adventitia or serosa, which consists of connective tissues that separate the intestine from the surrounding peritoneal cavity.
1.4.1.1 Cell junctions
Intestinal epithelial cells (IECs) are tightly packed together in a single layer that prevents passage of bacteria to the underlying tissues. At the same time, the epithelium remains semi-permeable to allow passage of nutrients and ions from the lumen to the IECs and underlying tissues. Transport can take place both through (intracellular) and between (paracellular) IECs. The IECs are therefore connected to one another with the help of protein complexes, which provide layers of cell dynamic junction structures, referred to as the epithelial junction complex.
The junction complex comprises tight junctions (TJ; located closest to the lumen), followed by adherence junctions (AJs), and desmosomes. TJs are intercellular complexes made up of transmembrane proteins, such as claudins and occludins that prevent paracellular leakage14. AJs consist of transmembrane
proteins, such as E-cadherin and catenin proteins15. Both AJs and desmosomes
are located beneath the TJs and are responsible for strengthening cell-to-cell adhesion. Together, these junction structures act as a selective physical barrier. In addition, several components of luminal contents, including dietary
9
components and stimuli from the microbiota can act beneficially on IECs by affecting tight junction structures16,17.
1.4.1.2 Intestinal epithelial cell types
The IECs includes several cell types. These are derived from stem cells, which are located at the bottom of the intestinal crypts. Daughter cells to the stem cells proliferate before they differentiate into highly specialized and differentiated IECs18. IECs consist of two classes: absorptive and secretory enterocytes. The
secretory type can be further subdivided into four populations: mucus-producing goblet cells, antimicrobial peptide-mucus-producing Paneth cells, hormone-secreting enteroendocrine cells, and chemical-sensing Tuft cells.
Enterocytes are the predominant absorptive cell type in the intestine villi. One of their primary functions is to absorb digested nutrients. They have typical characteristics, such as dense microvilli, which further increase the absorptive area towards the lumen. The cell membrane of the microvilli contains enzymes needed to digest dietary components, and membrane-bound glycoproteins, like transmembrane mucins, which constitute the glycocalyx. Enterocytes are also capable of producing chemokines in the event of infection or epithelial damage19. Furthermore, enterocytes in both SI and LI constantly express
antimicrobial peptides, such as β−defensins 120,21.
Paneth cells mainly resides in the SI and unlike enterocytes, they are long-lived cells (about two months). They are located at the bottom of the crypts, juxtaposed with the stem cells and function as nursing cells by providing growth factors to the stem cells22. Paneth cells are characterized by their
apically-oriented secretory granules in the cytoplasm, containing antimicrobial proteins, including defensins and lysosome. Defensins are bactericidal and anti-viral components, as they can disrupt bacterial membranes or target the viral envelopes23,24. The high amounts of various antimicrobial proteins produced by
Paneth cells help to guard the intestinal crypt and create an almost sterile local environment in the crypt lumen25. Altered expression of defensins is associated
with inflammatory bowel diseases (IBDs), like Crohn’s disease21,26
Goblet cells are large mucus-producing cells, interspersed between enterocytes, located both in the crypts and at the villus/luminal compartments. They are named goblet, because they contain mucin-containing vacuoles that shape the cytoplasm, which is reminiscent of the structure of a goblet. In contrast to Paneth cells, the frequency of mucus-producing goblet cells progressively increases along the GI being highest in the most distal part of the intestine. The entire intestinal surface is covered by mucus with the gel-forming mucin (MUC) 2 as its main constituent. The small intestine has one layer of mucus that
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is relatively loose, while the colon has a thicker two-layered system with an inner and an outer mucus layer27. Goblet cells also express the protease
resistance trefoil factor 3 (TFF3), which interacts with MUC2 and influences the mucus viscosity28. The intestinal mucus layer has several important functions,
which include: lowering antigen exposure to the immune system, protecting the epithelium from self-digestion by endogenous enzymes, and providing matrix for secreted components of both the innate immune system, such as AMPs and adaptive immune system, such as secretory IgA (sIgA)27. Goblet cells may also
be induced to secrete mucin in response to inflammatory conditions29,30.
Enteroendocrine cells, are hormone-producing cells located throughout the whole intestinal tract31. They produce immunologically active factors, such as
substance P, a mediator of inflammation32-34. They are classified into several
types, depending on their hormone production. Their main function is to sense changes in the luminal content and secrete neuropeptides and hormones into adjacent capillaries and regulate various digestive functions35,36.
Tuft cells are a rare type of cells, characterized by long microvilli projections and deep surface invaginations into the intestinal epithelium with as yet unknown functions. They are proposed to act as chemical sensors for the luminal contents. Recently, it was shown that tuft cells are involved in immune responses in parasitic infections37.
In addition to enterocytes, the intestinal epithelium contains microfold cells (M cells), found in the follicle-associated epithelium (FAE) of subepithelial lymphoid aggregates that lie in the mucosa and submucosa. M cells have a flat apical surface and are specialized for the uptake and transport of antigens from the lumen into the underlying tissue for further presentation to the adaptive immune system.
1.4.1.4 Microbiota
The biogeography of the microbiota is still being investigated. However, the bacterial communities seem to differ dramatically between intestinal compartments from the stomach to the rectum. In the SI, the bacterial load is several order of magnitude lower, compared to the colon, probably due differences in the nutritional and biochemical milieu. Shorter transit time, as well as antimicrobials are factors that limit bacterial growth in the SI38. In
contrast, conditions in the colon, promote a dense and diverse community of bacteria, such as anaerobes with the ability to utilize undigested carbohydrates. The intestinal microbiota of an individual consists of tens of thousands of species, dominated mainly by the phyla Bacteroidetes and Firmicutes, Proteobacteria and Actinobacteria39. Studies have shown that the
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and is dominated by two bacterial genera, namely Streptococcus and Neisseria39. Microbiota can be affected by diet and environmental factors,
although despite fluctuations, the microbiota has been shown to be relatively stable within the individual40.
In addition to production of important nutrients, such as vitamins and short-chain fatty acids (SCFAs), the microbiota is essential for the function of the
immune system41. Studies in germ-free animals have demonstrated the
importance of gut microbiota in the development and regulation of innate and adoptive immune responses. For example, germ-free mice have less developed gut-associated lymphoid tissue structures and reduced levels of sIgA compared to conventionally raised mice42. By occupying biological niches within mucosal
areas, the microbiota also take part in the prevention of invasion of pathogenic bacteria43.
1.4.2 Tissues of the mucosal immune system and intestinal immune cells
1.4.2.1 Gut-associated lymphoid tissue (GALT)
Organized lymphoid tissue in the gut is referred to as the GALT. It constitutes the largest immunological organ in the body. The GALT includes Peyer’s patches (PPs), the appendix and solitary intestinal lymphoid tissues (SILTs). SILTs consist of dynamic microscopic lymphoid aggregates ranging from small cryptopatch-like structures to large, mature isolated lymphoid follicles (ILFs)44.
PPs are localized in the SI and have developed before birth, while SILTs are located along the GI tract and develop early after birth, which is considered to be due to stimulation by the microbiota45.
The best studied components of GALT are the macroscopically visible PPs. The size and density of PPs increase from the jejunum to the ileum and they are particularly concentrated in the distal ileum46. Each PP consists of at least five
aggregated lymphoid follicles. A follicle can be divided into three main domains. These domains include the follicular area, the interfollicular area, and the FAE47. The follicular area has a germinal center (GC), and contains primarily
proliferating B lymphocytes, which when matured to plasma cells, are the major source of intestinal IgA48,49. The follicle is flanked by the interfollicular domain,
which is rich in T cells. The FAE is separated from the follicle by the subepithelial dome (SED) region, which lies directly beneath the FAE and is rich in APCs and lymphocytes. DCs in the SED region have been shown to extend their dendrites into the gut lumen and sample antigens50.
12 1.4.2.2 Lamina propria immune cells
The LP and the epithelium are the effector sites of the intestinal immune system. The LP contains T cells, B cells and plasma cells together with numerous types of innate immune cell populations, including ILCs, DCs, macrophages, and occasional eosinophils and mast cells.
Both CD4+ T cells and CD8+ T cells with effector memory phenotype are
abundant in LP, while γδ T cells are rarely found. The CD4+ T cell compartment
of LP is highly diverse and contains several subsets, such as Th1, Th17 and Tregs51. Furthermore, LP contains plasma cells, which constitute the basis for
the secretory sIgA52. IgA are recognized by polymeric immunoglobulin receptors
on the basolateral surface of IECs, which shuttle IgA dimers across the cells through a process called transcytosis and release sIgA53. Plasma cells also
secrete β-defensins and thereby contribute to the protection from invasion by bacteria into the LP54. ILCs are found in LP with important roles in intestinal
immunity, inflammation and GALT development11. Intestinal DCs have a key
role in maintaining homeostasis in the gut55. As such, they produce factors that
favor tolerogenic responses, which promotes differentiation of Treg cells56.
Macrophages in the gastrointestinal mucosa represent the largest pool of tissue macrophages in the body57. During non-pathological conditions, macrophages
have inflammation-suppressive phenotypes and thus contribute to homeostasis in the gut58. Eosinophil and mast cells are also present in normal intestinal
mucosa and have important physiological roles, such as tissue repair and epithelial integrity as well as influencing peristalsis51.
1.4.2.3 Intraepithelial lymphocytes
The intestinal epithelium contains numerous T cells that are located between enterocytes. The T cells are of different phenotypes and are distributed differently in the SI and the LI1,59. As such, IELs are more frequent in SI where
they comprise 15-20% of the cells in the epithelium1. The various cell
populations of IELs, include conventional αβ T cells with coreceptors CD4 or CD8 and unconventional T cells including TCRαβ coreceptor-negative (or double-negative, DN) T cells and γδ T cells1,60. Conventional CD8+ T cells
dominate in the SI, while in the LI there is an almost equal proportion of CD8+
and CD4+ T cells and DN T cells1. γδ T cells are about 10% of IEL in both SI and
LI1. Most IELs are antigen experienced T cells with effector memory phenotype. The majority of IELs, regardless of other cell surface molecules, express CD103, also known as αE integrin, which anchors IELs to the epithelial cells61. Many
IELs have cytolytic capabilities characterized by the presence of cytotoxic granulae in the cytoplasm62. Additionally, they have other functions, like
induction and maintenance of oral tolerance and surveillance of the IECs’ functions60.
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1.6 Pattern recognition receptors
The basis for innate immunity relies on pattern recognition receptors (PRRs). PRR are proteins specialized in recognizing evolutionarily conserved structures of components in microbes, known as microbial associated molecular patterns (MAMPs). Most PRRs can also be activated by non-microbial and endogenous signals, which are generated during tissue damage, known as damage-associated molecular patterns (DAMPs). PRRs can be classified into several families based on protein domain homology. Examples are Toll-like receptors (TLRs), nucleotide binding domain, leucine-rich repeat (LRR)-containing receptors (NLRs), RIG-I like receptors (RLRs) and C-type lectin receptors (CLRs). Activation of PRRs leads to production of innate responses that help the host to remove the threat and restore tissue homeostasis63. This includes
induction of pro-inflammatory cytokines and regulatory miRNA (described more in Chapter 1.9). Further, PRR activation can lead to cellular responses, such as induction of phagocytosis, autophagy and/or cell death64,65.
PRRs are strategically located in different subcellular compartments. PPRs can be either membrane-bound at the cell surface and at the endosomal membranes or occur free in the cytoplasm. For instance, TLRs and CLRs are found at cell surfaces or in association with endocytic compartments, while NLRs and RLRs are mainly located in the cytoplasm, where they survey for the presence of intracellular pathogens. This distribution of PRR is meant to effectively detect different types of threats and to prevent inappropriate activation. The multiplicity and diversity MAMPs present on microbes and the recognition by numerous PRRs, trigger different signaling pathways and contribute to the complexity and magnitude of innate responses. In addition to sensing danger, PRR also play an important role in the maintenance of epithelial barrier integrity, production of antimicrobial proteins (such as defensins) and transcytosis of IgA to the gut lumen63.
1.6.1 Toll-like receptors
The best-known PRRs are members of the TLR family. They are transmembrane glycoproteins characterized by an extracellular N-terminal LRR motif, which recognize ligands, and a cytoplasmic Toll/IL-R homology (TIR) domain that initiates the intracellular signaling pathways in response to receptor activation66. TLRs can recognize a broad range of microbes, such as bacteria,
virus, fungi and parasites. Depending of the type and location of TLRs, different regulatory signaling pathways are activated.
There are 10 identified TLRs in humans. TLRs located to within cell membranes (TLR1, TLR2, TLR4, TLR5, TLR6 and TLR10) mainly recognize bacterial products, while TLR, usually located in intracellular membranes (TLR3, TLR7,
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TLR8 and TLR9), appears to be suited for viral detection and recognize nucleic acids66.
Each receptor shows specificity by recognizing distinct bacterial MAMPs. For instance, TLR4 recognizes lipopolysaccharide (LPS), a molecule found in the outer membranes of Gram-negative bacteria67. Activation of TLR4 further
involves the accessory molecules myeloid differentiation protein 2 (MD-2) and LPS-binding-protein (LBP) as well as CD14, which bind LPS and act as a co-receptor68,69. TLR5 senses bacterial flagellin, which is the structural component
of the flagellum, a locomotor organ that is mostly associated with Gram-negative bacteria70. TLR2 plays a role in recognition of peptidoglycan (PGN) and
lipoproteins71. TLRs occur as homo- or heterodimer in different combinations of
TLRs, which further increase the variety of PAMPs that can be recognized. For instance, TLR2 can form a heterodimer with either TLR1 or TLR6 and recognize lipoprotein with different lipid moieties72. TLR3 is a receptor for viral dsRNA73,
while TLR7 and TLR8 recognize ssRNA from RNA virus74-76. TLR9 primarily
recognize unmethylated CpG DNA, which is uncommon in the mammalian genome77.
1.6.1.1 Toll-like receptor signaling pathways
There are two well-studied signaling pathways in TLR activation, which depend on the type of TIR-adaptor protein being recruited after receptor activation. The myeloid differentiation primary response gene 88 (MyD88) is the commonly used TLR adaptor for initiating signal cascades, also referred to as the MyD88-dependent signaling pathway. Following activation, MyD88 oligomerizes to form a large signaling platform, which includes several MyD88 molecules, TIR domain–containing adapter protein (TIRAP) and members of the IRAK family proteins. This complex assembles with TNF receptor–associated factor 6 (TRAF6)78 and through further intracellular molecular reactions, NF-κB is
activated, which drives the transcription of several genes involved in innate
immune responses79. For TLR7, TLR8 and TLR9, the MyD88-dependent
signaling can also result in the expression of type I interferons (IFNs), which are important in controlling viral infections80. The other signaling pathway is
referred to as the MyD88-independent/TRIF-dependent pathway, which is used
by TLR3 or TLR481 and leads to induction of members of the interferon
regulatory transcription factor (IRF) family, responsible for the induction of IFNs82.
1.6.2 Other families of pattern recognition receptors
NLRs (Nucleotide-binding oligomerization domain-like receptors) are cytosolic sensors with more than 30 members. They are characterized by the presence of a C-terminal LRR domain, a central NLR, and an N-terminal domain
15
responsible for signal transduction. These receptors are expressed in many cell types, including immune cells, epithelial cells and endothelial cells. NLRs are divided in subfamilies based on the N-terminal-containing domain, which can mediate signaling, either with the caspase recruitment domain (CARD) or the putative protein-protein interaction (PYRIN) domain. NOD1 and NOD2 are the most studied receptors within this family and can sense components of bacterial outer membranes or cell walls. NOD1, specifically recognizes the peptidoglycan moiety of Gram-negative bacteria, named γD-glutamyl-meso-diaminopimelic
acid (iE-DAP)83. NOD2 recognizes the peptidoglycan fragment
muramyl-dipeptide (MDP), that is present in the peptidoglycan of both Gram-negative and Gram-positive bacteria84.
NLRPs (NACHT, LRR and PYD domains-containing proteins) are one
subfamily of NRLs that contain a PYRIN, instead of a CARD domain. They are involved in the formation of a multiprotein complex, referred to as the inflammasome, which initiate innate immune responses characterized by the secretion of pro-inflammatory cytokines IL-1β and IL-1885. Inflammasome
formation is considered to be an important host response to a wide range of diseases, such as inflammatory diseases, cancer, metabolic and autoimmune disorders86.
RLRs (Retinoic acid-inducible gene-I-like receptors, or RIG-I-like receptors) are DEXD/H box RNA helicases that detect the presence of foreign RNA within the cytosol of the cell. They are situated at specific locations within the cytosol where viral- entry or replication can occur and recognize parts of the viral genome components formed as replicative intermediates during viral growth. The RLRs generate activation signals that drive IFNs production and elicit an intracellular immune response to control virus infection87. There are currently
three members of RLRs: retinol acid-inducible gene I protein (RIG-I), melanoma differentiation associated gene-5 protein (MD5) and laboratory of genetics and physiology protein (LGP2)88-90.
CRLs (C-type lectin receptors) are a family of membrane-associated receptors, which recognize the carbohydrate structures present on pathogens. There are currently, more than 60 identified CLRs in humans. The extracellular portion of CLR consists of a calcium-dependent carbohydrate recognition domain (CRD). These receptors are widely recognized to play an essential role in antifungal immunity91.
1.6.3 Pattern recognition receptors in human intestine
IECs are normally the first to detect the presence of pathogens or secreted bacterial factors in the gut lumen and respond immediately by producing a
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variety of mediators, depending on the type of stimuli. Therefore, IECs represent an important site of crosstalk between the immune system and the microbiota. Members of the following families, TLRs, NLRs, RLRs and CRLs
have been detected in the intestinal mucosa92. To avoid unappropriated
stimulation, PRR activation is tightly regulated in several ways.
PRRs are generally present at low levels in IECs. For instance, TLR4 as well as its co-receptor MD2 are expressed at low levels in IECs, which is thought to explain the relative hypo-responsiveness to PAMPs, such as LPS93. However,
during pathological conditions, for instance in IBD, TLR4 expression is
up-regulated94. Similarly, NLRP2, NLRP6 and NLRP8 are up-regulated in
autoimmune conditions such as celiac disease95.
Expression of TLRs are generally restricted to the basolateral surface of IECs, such as for TLR5, where it can trigger production of cytokines in response to flagellin that has reached the internal tissue side of the epithelium96. The spatial
regulation of this TLR, is thought to ensure that an immune response is only mounted when bacteria have breached the host epithelial layer63. Thus, the
consequences of PRRs signaling depend on the site of the cell where the ligand is encountered. It has been shown that, TLR activation through apical or basolateral surface domains produces distinct transcriptional responses. For example, basolateral exposure of IECs to TLR9 ligands results in activation of NFκB, while apical stimulation results in an opposite, inhibitory signaling cascades and further provides intracellular tolerance to subsequent TLRs
challenges97. Similar mechanisms may also explain why challenges with
bacterial components can differ depending on whether challenge with bacteria are done using cells in ordinary tissue culture compared to differentiated polarized tight monolayers98
The microbial ligands recognized by PRRs are not unique to pathogens but are also expressed by commensal microorganisms. The commensal bacteria are under normal conditions recognized by PRR, such as TLRs and NODs and this interaction plays a crucial role in the maintenance of intestinal epithelial homeostasis99,100. Several protective features following PRR activation have
been demonstrated, which include epithelial cell proliferation, maintenance of tight junctions and induction of antimicrobial peptides100-103. By which
mechanisms commensal bacteria modulate inflammatory responses in the gut is unclear. Meanwhile, the regulation of PRR expression and how these receptors are spatially distributed within the polarized IECs has been suggested to play a major role. Negative regulators of TLR signaling, such as expression of microRNA, is another mechanism employed by IECs that probably contributes to creating a tolerant environment within the intestine104.
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1.7 Cytokines
Cytokines are small soluble signalling proteins that function in cellular communications through ligand and receptor interactions. They are messengers in immune responses and can be produced by many cell types, such as immune cells and epithelial cells. Cytokines can act in an autocrine, paracrine or endocrine manner and therefore can produce both local and systemic effects. Binding to the corresponding receptor(s), they induce intracellular signaling, leading to cellular responses, which include activation, proliferation, and differentiation. Cytokines are pleiotropic and redundant molecules, meaning that the same cytokine can affect multiple cell-types and many cytokines can exert similar actions in the same target cell. They further function in both innate and adaptive immune responses, which further illustrates their complex biological actions.
Cytokines can be divided into families, based on target cells and proposed functions. Interleukins (ILs; cytokines that signal between immune cells), lymphokines (cytokines produced by lymphocytes), monokines (cytokines produced by monocytes/macrophages/DCs), colony stimulating factors (CSFs; cytokines involved in stem cell proliferation and differentiation), chemokines (cytokines with the capacity to attract immune cells), and interferons (IFNs; cytokines that interfere with virus infection) are well-described, partly over-lapping families of cytokines. Further type of classification is based on the outcome immune responses, i.e. whether the cytokine promotes or inhibits inflammation or acts on adaptive immune cells. Representative examples of pro-inflammatory cytokines are TNF-α, IL-1β, IL-6, IL-8 and IFN-γ. IFN-γ is a member of the IFN family and is a typical pleotropic cytokine promoting cell-mediated immune responses, increase in antigen presentation and expression of other pro-inflammatory cytokines105. Expression of pro-inflammatory cytokines
results in hallmarks of the inflammation phenotype, which include vasodilation, increased vascular permeability, and influx of immune cells, e.g. monocytes and neutrophils to the site of infection. In contrast, cytokines that suppress the activity of inflammation signaling are called anti-inflammatory cytokines. IL-10 and transforming growth factor-β (TGF-β) prevent host cell damage from excessive inflammation by counteracting the functions of pro-inflammatory cytokines106.
IECs are one of the important cellular sources for cytokine production. During infection several different cytokines are produced in order to mobilize the immune system and eliminate the infectious agents. After the elimination and resolution of diseases, the expression of the cytokines is down-regulated and usually returned to normal baseline levels. Therefore, aberrant expression of cytokines, often long-standing production, can give signals that can sustain
18
pathophysiological processes. For example, increased production of IL-1β, IL-6, IL-8, TNF-α and CCL20 are associated with several inflammatory disorders, such as IBD and ongoing infiltration of immune cells to the site of inflammation107. Therefore, an understanding of how pathogens can affect
cytokine production at sites of infection may explain some of the pathological outcomes in many infections or inflammatory diseases.
1.7.1 Interleukin-1β and Interleukin-18
IL-1β and IL-18 are members of the IL-1 family, currently comprising 11
cytokine members107. IL-1β and IL-18 are considered pro-inflammatory
cytokines and are produced by various cell types, including macrophages, neutrophils and epithelial cells108. IL-1β is mainly induced in response to
microbial recognition, mediated by activated PRR, like TLRs and NODs109.
IL-18, on the other hand is constitutively expressed in nearly all cells in healthy tissues110. Members of the IL-1 family, like IL-1β and IL-18 are typically
synthesized as precursors and must be cleaved by enzymes, for example caspase-1 (CASP1), to become active secreted cytokines111. The secretion of IL-1β
and IL-18 are part of the inflammasome forming response, which is activated by pathogens and are responsible for the activation of crucial inflammatory responses85.
There are two types of receptors for IL-1: IL-1 receptor type 1 (IL-R1) and IL-1 receptor type 2 (IL-1R2). The heterodimeric complex of IL-1R1 is associated with an IL-1R accessory protein (IL-1RAcP), which together constitutes the functional receptor for IL-1112. IL-1R has a long and conserved cytoplasmic
Toll/IL-1R (TIR) domain, while IL-R2 has a short intracellular domain and acts as a suppressor of IL-1 activity, supposedly by competing for IL-1 binding109.
The receptor for IL-18 (IL18R) consists of IL-18 receptor α−chain (IL-18Rα), and the IL-18 co-receptor, termed IL-18 receptor β−chain (IL-18Rβ), which together are required for effective signaling113. Following binding, the complex
of IL-18 with the IL-18Rα and IL-18Rβ is similar to that formed by IL-1β and the accessory protein IL-1RAcP107. The activity of IL-18 is balanced by the
presence of an inhibitor, called IL-18 binding protein (IL-18BP), which is secreted mainly by immune cells upon pro-inflammatory response to inactivate IL-18. Similar to IL-R2, IL-18BP is considered a decoy receptor that ensures a balance between the amplification of innate immunity and the regulation of cytokine signaling114.
The signal transduction for IL-1β and IL-18 share common features and are mediated through IRAK family members and TRAF6, which activate
mitogen-19
activated protein kinases (MAPKs) and the transcription factor NF-κB, resulting in expression of pro-inflammatory cytokines107.
1.7.2 Tumor Necrosis Factor−α
TNF-α was initially identified as a factor responsible for necrosis of certain tumor cells115. Now, it is known that TNF-α is a potent inflammatory mediator
and plays a central role for induction of inflammation, including cytokine production, expression of adhesion molecules and growth stimulation 116. The
multitude of functions elicited by TNF-α makes it one of the most pleiotropic mediators in immune responses107. TNF-α is a classical member of the TNF
superfamily of proteins, which includes 30 receptors and 19 associated ligands with diverse functions in cell differentiation, inflammation, and apoptosis117.
TNF-α is mainly secreted by activated macrophages, however other cell types, including, T cells, mast cells and epithelial cells can also produce TNF-α118.
TNF-α is synthesized as a transmembrane precursor, arranged in homodimers at the cell membrane. To generate the soluble form, the cytokine is released by proteolytical cleavage by proteases, like the TNF-α converting enzyme (TACE)118. Both the soluble and the cell membrane-bound form of TNF-α are
active and have distinct effects in innate responses119. The protease TACE is
further involved in the processing of several other cell membrane-associated receptors and can execute the release of soluble forms of receptors, such as TNF-α receptors, which can neutralize the action of TNF-α and thus limit cytokine availability to other cells120.
The receptors for TNF-α are TNF receptor type 1 (TNFR1) and TNF receptor type 2 (TNFR2). The two TNFRs have been reported to mediate distinct biological effects. As such, TNFR2 mediate signals promoting tissue repair and angiogenesis121, while TNFR1 are suggested to support the pro-inflammatory
effects of TNF-α107.
1.7.3 Chemokines
Chemokines are mainly produced to recruit leukocytes of both innate and adaptive immunity to inflammatory sites. There are 44 chemokines and 23 chemokine receptors in the human genome122. Chemokines are a group of small
(8–12 kDa) peptide mediators, classified into four families, based on the positioning of the N-terminal cysteine residue. The four families are: C-, CC-, CXC- and CX3C- families, where C represent the number of N-terminal region cysteine residues and X represents the number of intervening amino acids123.
Chemokine signals mainly through binding to members of the G protein– coupled receptors (GPCR) superfamily124.
20
The CC family is characterized by cysteine residue located adjacent to one another. The CC chemokine ligand 20 (CCL20) is an example of one such chemokine. CCL20 is a small chemokine of about 8 kDa that is constantly expressed at low levels by epithelial cells in various peripheral tissues125.
Immune cells such as activated neutrophils and lymphocytes can also produce this chemokine. Only one receptor, CC receptor 6 (CCR6) for CCL20 is known. The CCR6 receptor is found prominently on immune cells, such as DCs, B cells and T cells, including T cell subsets Th17 and Treg126. In the literature, this
interaction is referred to as the CCR6–CCL20 axis, and is implicated in several autoimmune diseases, including IBD127,128.
CXC cytokines can be further subdivided based upon the presence or absence of the amino acid sequence, glutamic acid-leucine-arginine (the ELR motif), prior to the first cysteine. The ELR+ CXC chemokines are: CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, CXCL7, and CXCL8/IL-8). They act mainly through CXCR2 and attracts neutrophils and possess angiogenic properties. Some of the ELR−
CXC chemokines are: CXCL4, CXCL9, CXCL10 and CXCL11, which act mainly through CXCR3 and attract lymphocytes, like Th and NK cells and harbors angiostatic properties129.
Interleukin-8 (IL-8) or CXCL8 is one of the first chemokines to be isolated (1987) and is the most studied chemokine130. Originally, IL-8 was named
neutrophil chemotactic factor (NCF), because this chemokine mainly attracted neutrophils to sites of infection131. IL-8 can also attract several types of immune
cells and is considered a key mediator of the inflammation response132. There
are several receptors for IL-8. The most studied are the two GPCRs; CXCR1 and CXCR2133. Following chemokine binding to the receptor, intracellular signaling
cascades are activated within target cells, such as neutrophils and induce several cell responses, including chemotaxis and/or degranulation of neutrophil
contents134. In addition, IL-8 signaling induces expression of adhesion
molecules in target cells, such as the lymphocyte function-associated antigen 1 (LFA-1) and the macrophage-1 antigen (Mac-1), which are required for chemotaxis135.
CX3CL1 is a member of the CX3C chemokine family. This cytokine is mainly produced as a cell membrane protein. Once cleaved from the cell surface it functions as a soluble chemoattractant. CX3CL1 attracts predominantly T and B cells, NK cells, and monocytes via chemokine receptor CX3CR1136.
